Discordant Umbilical Cord Drug Testing Results in Monozygotic Twins.

Our laboratory received segments of umbilical cord that originated from identical twins for routine toxicology analysis. The specimens were analyzed multiple times by liquid chromatography tandem mass spectrometry. The umbilical cord from newborn #1 was positive for hydromorphone only (1.06 ng/g), and the umbilical cord from newborn #2 was positive for hydromorphone (0.81 ng/g) and benzoylecgonine (5.41 ng/g). The hydromorphone results are consistent with maternal administration of hydromorphone; however, the cause of the discrepant benzoylecgonine results in the umbilical cords from the identical twins is unknown.

Plasma Concentrations of Fentanyl Achieved With Transdermal Application in Chickens.

Providing appropriate analgesia is an important concern in any species. Fentanyl, a µ-receptor specific opioid, use is common in mammalian species but has been incompletely evaluated for this purpose in avian species. Transdermal fentanyl patches were applied to domestic chickens (n = 10) of varying breeds for 72 hours. Repeated blood samples were collected from the birds to assess time-concentration of fentanyl and norfentanyl in plasma, as assayed by liquid chromatography-mass spectrometry, throughout patch application and for 48 hours after patch removal. Compartmental modeling was used to characterize the elimination profiles. Evaluation as a large bolus, followed by slower elimination rates over the remaining time, best fit the data as a one-compartment open model. Although maximum plasma fentanyl concentrations varied substantially by individual birds, chickens trended into 2 general groups of maximum plasma concentration, clearance, and volume of distribution, which was attributed to absorption variability. For all birds, harmonic mean of elimination half-life was 7.2 ± 3.7 hours and showed less individual variation than the other pharmacokinetic parameters. Because the application of transdermal fentanyl patches in the chickens achieved plasma fentanyl concentrations considered therapeutic in people, this approach could provide an additional analgesic option for avian patients.

Determination of methamphetamine enantiomer composition in human hair by non-chiral liquid chromatography-tandem mass spectrometry method.

Chiral separation is crucial for investigating methamphetamine positive cases. While (S)-(+)-enantiomer of methamphetamine (S-MAMP) is a schedule II controlled substance, (R)-(-)-enantiomer (R-MAMP) is an active ingredient of a few over-the-counter drugs in the United States. Among biological specimen types, hair provides greater detection window than blood, urine or oral fluid, and are therefore regarded with particular interest. Herein we describe a novel non-chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to directly determine methamphetamine enantiomeric composition (percentage) in hair specimens. Hair samples were washed once with acetone, powdered, incubated overnight at 53°C in 0.1M hydrochloric acid (HCl), and subjected to a solid phase extraction (SPE). The extracts were derivatized using Marfey's reagent at 53°C for 60min. The final mixture was analyzed by LC-MS/MS. Chromatographic separation was achieved using a C18 Kinetex analytical column and 60% (v/v) aqueous methanol as mobile phase (isocratic). Triple quadrupole mass spectrometer was equipped with an electro-spray ionization (ESI) source operating in negative mode and the chromatograms were acquired using a multiple-reaction monitoring (MRM) approach. The results were expressed as ratio of R- to S-MAMP and then derived to composition percentages without requiring quantitating each enantiomer. The method was precise and accurate across 0-100% S-composition at a range of 80-18,000pg/mg. The performance of the new method was compared with an (S)-(-)-N-trifluoroacetylprolyl chloride (S-TPC) derivatization and gas chromatography-mass spectrometry (GC-MS) method on authentic methamphetamine-positive hair samples. Not only the new Marfey's reagent approach presented satisfactory correlation with the S-TPC approach, but it also exhibited significantly improved quality (e.g., S/N) of the chromatograms. In summary, our protocol employs cost effective and minimally hazardous Marfey's reagent to derivatize trace amounts of methamphetamine extracted from hair samples and a non-chiral LC-MS/MS approach to separate and identify the two enantiomers. The method allows determination of the methamphetamine enantiomeric composition without requiring quantitation of each enantiomer and is therefore well suited for further investigate previously determined methamphetamine positive cases. This method represents a viable tool for evaluation of long-term drug exposure.

Detection of Drugs in Nails: Three Year Experience.

Nails (fingernails and toenails) are made of keratin. As the nail grows, substances incorporate into the keratin fibers where they can be detected 3-6 months after use. Samples are collected by clipping of 2-3 mm of nail from all fingers (100 mg). We present drug testing results from 10,349 nail samples collected from high-risk cases during a 3-year period of time. Samples were analyzed by validated analytical methods. The initial testing was performed mostly using enzyme-linked immunosorbent assay, but by liquid chromatography-tandem mass spectrometry (LC-MS-MS) as well. Presumptive positive samples were subjected to confirmatory testing with sample preparation procedures including washing, pulverizing, digestion and extraction optimized for each drug class. The total of 7,799 samples was analyzed for amphetamines. The concentrations ranged from 40 to 572,865 pg/mg (median, 100-3,687) for all amphetamine analytes. Amphetamine and methamphetamine were present in 14% of the samples, 22 samples were positive for 3,4-methylenedioxymethamphetamine (0.3%), 7 for methylenedioxyamphetamine (0.09%) and 4 for 3,4-methylenedioxy-N-ethylamphetamine (0.05%). Cocaine and related analytes were found in 5% samples (7,787 total), and the concentration range was 20-265,063 pg/mg (median 84-1,768). Opioids overall ranged from 40 to 118,229 pg/mg (median 123-830). The most prevalent opioid was oxycodone (15.1%) and hydrocodone (11.4%) compared with 1.0-3.6% for the others, including morphine, codeine, hydromorphone, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and oxymorphone. Carboxy-Δ-9-tetrahydrocannabinol positivity rate was 18.1% (0.04-262 pg/mg, median 6.41). Out of 3,039 samples, 756 were positive (24.9%) for ethyl glucuronide (20-3,754 pg/mg, median 88). Other drugs found in nails included barbiturates, benzodiazepines, ketamine, meperidine, tramadol, zolpidem, propoxyphene, naltrexone and buprenorphine. Nail analyses have become a reliable way of determining the long-term use and abuse of drugs. Extraction techniques are simple and produce accurate and precise results. Sensitive analytical instrumentation, mainly LC-MS-MS, allows for detection of femtogram (10(-15) g) quantities of substances in nails. Samples were from a high-risk population, therefore the extraordinary positivity rate was observed.

Confirmation and quantification of clenbuterol in horse urine using liquid chromatography tandem mass spectrometry triple quadrupole.

Clenbuterol (CLE) is used in horses as a bronchodilator and for its anabolic steroid-like effects. CLE is a Class 3 drug according to current Association of Racing Commissioners International (ARCI) Uniform Classification Guidelines. The Racing Medication and Testing Consortium recommended a urine CLE threshold of 140 pg/mL after careful scientific review of the results of studies describing the disposition of CLE in the horse and this threshold was adopted by the ARCI. Enzyme-linked immunosorbent assay was previously used to screen samples for CLE in Illinois, but could not detect such low concentrations in urine. Thus, a liquid-liquid extraction of CLE from urine followed by quantification by liquid chromatography-tandem mass spectrometry was developed and validated. Method validation included testing stability, ion suppression and enhancement, precision, accuracy and uncertainty. Intra-, interday and total precision and accuracy were calculated for each control and found to be within the ±15% acceptance range. The Guide to the Expression of Uncertainty in Measurement approach was used to calculate uncertainty, which was 11% at the 95% confidence level. In the past 5 years, only 15 samples were reported as positive for CLE in Illinois. This new method was used in a pilot program to screen and confirm samples received from thoroughbred and harness horses.

Equine total carbon dioxide testing in Illinois in 2012.

During prolonged strenuous exercise, racehorses can experience acidemia. To counteract this phenomenon, trainers can administer blood alkalizing agents that raise the plasma pH and total carbon dioxide (TCO2) concentration. In Illinois, the administrative threshold for TCO2 in plasma is 37.0 mmol/L. Because accuracy in the reported measurement of TCO2 must be ensured, uncertainty measurements are often issued alongside the reported concentrations. We report a validated method for measuring TCO2 levels in equine plasma using the Beckman UniCel DxC 600. A six-point calibration curve ranging from 5 to 50 mmol/L is analyzed along with controls at four TCO2 levels with each set of samples. Using this method, we collected data from 5,199 race samples during 2012, with 134 being from thoroughbred horses and 5,065 from standardbred horses. During method validation, uncertainty was determined using the simplified Guide to the Expression of Uncertainty in Measurement approach and was found to be 3% at 99.7% confidence level with eight measurements. Additionally, to investigate other variables that could have an effect on TCO2 levels, we collected the gender, breed, Lasix(®) status, strong ion concentration, pre- or post-race collection time and track location of all horses tested during that year. The samples had an overall mean TCO2 concentration of 30.5 ± 2.0 mmol/L. The other physiological and environmental data were analyzed using analysis of covariance tables. These results indicate gender, breed, furosemide status, collection time and track location to be strongly correlated (P < 0.0001) to TCO2 levels. Thoroughbred status was found to have no effect. Finally, TCO2 concentrations were highly correlated (P < 0.0001) to sodium and chloride ion concentrations. No correlation was found between TCO2 and potassium concentrations. The results show that there are several environmental and physiological factors that can affect TCO2 concentrations. The concentration of other strong ions present in the blood may indicate doping status.

Dependence of mass spectrometric fragmentation on the bromine substitution pattern of polybrominated diphenyl ethers.

This study investigates the link between the bromine substitution and the mass spectrometric fragmentation of polybrominated diphenyl ethers (PBDEs). The mass spectra of 180 PBDEs were obtained in both electron impact (EI) and electron capture negative ionization (ECNI) modes using a single quadrupole mass spectrometer (MS) as well as EI using a tandem MS (MS/MS). The major ions are M(+), [M-2Br](+), [M-2Br](2+) and [M-nBr-28](+) in EI, and Br(-), [HBr2](-) and [C6BrnO](-) in ECNI. In EI-MS, congeners without ortho bromine or having 2,3 substitution on one ring and no ortho bromines on the other were more robust than the others in each homolog. These congeners generated low [M-2Br](+) but relatively high [M-2Br](2+) in EI-MS and negligible [HBr2](-) in ECNI-MS. In EI-MS/MS, the molecular ions of these congeners required higher collision energy to debrominate, and produced additional ions of [M-nBr](+) and [M-nBr-28](+). Full ortho substitution promotes C-O cleavage forming [C6BrnO](-) in ECNI for congeners with >5 bromines. The relationship between the abundance of M(+) and collision energy of the EI-MS/MS was well characterized with a logistic regression model. Principle component analysis found associations between the inflection point collision energy and a few molecular descriptors. Quantum chemistry simulations revealed different EI-induced fragmentation mechanisms among four dibrominated congeners, supporting the hypothesized formation of a stable dibenzofuran-like intermediate during the fragmentation of some congeners but not of others.

Quantitation of fluphenazine in equine serum following fluphenazine decanoate administration.

Fluphenazine, a potent antipsychotic used to treat schizophrenia in humans, is used in racehorses as a performance-enhancing drug, and for that reason it has been banned by the Association of Racing Commissioners International. A liquid chromatography-tandem mass spectrometry method for detecting and quantitating fluphenazine in equine serum was developed and validated. The method was then employed to quantitate fluphenazine in serum samples collected from three study horses after intramuscular injection of fluphenazine decanoate. Stability testing showed that fluphenazine is stable in unextracted and processed samples as well as samples that have been subjected to up to three freeze-thaw cycles. The limit of detection and lower limit of quantitation of fluphenazine were determined to be 0.05 and 0.1 ng/mL, respectively. Precision was evaluated based on one-way analysis of variance of replicate quality control samples and was determined to be 27.2% at the 0.2 ng/mL level and 18.1% at the 2 ng/mL level. Bias was determined to be 0.55% at the 0.2 ng/mL level and 3.66% at the 2 ng/mL level. In two of three horses, fluphenazine was detected in serum up to 14 days post-administration. The highest detected concentration of fluphenazine in serum was 1.4 ng/mL.

Quantification of several acidic drugs in equine serum using LC-MS-MS.

The use of nonsteroidal antiinflammatory drugs in racehorses is allowed under most jurisdictions. Furosemide is administered to treat exercise-induced pulmonary hemorrhage. To help distinguish between therapeutic and illegal uses, racing regulatory bodies have set thresholds in serum for several drugs. The method for the simultaneous detection and quantification of furosemide, flunixin, ketoprofen, phenylbutazone and oxyphenbutazone using 500 µL of serum, and liquid extraction using diethyl ether : hexanes : dichloromethane followed by liquid chromatography tandem mass spectrometry quantitation, was developed and validated. Method validation included inter- and intraday precision and accuracy. Method validation also included bench-top, freeze-thaw, processed and long-term storage stability testing. For all stability testing, the compounds showed a breakdown of <15%. Inter- and intraday precision for all compounds was found to be within the acceptance interval of ±15% [±20% at the lower limit of quantitation (LLOQ)]. Accuracy data for all compounds were within the acceptance interval of ±15% (±20% at the LLOQ). Uncertainty was calculated using the simplified Guide to the Expression of Uncertainty in Measurement approach and was <30% for all drugs at 95% confidence level. The method was found to be both robust and accurate for all tested drugs.

Detection of various performance enhancing substances in specimens collected from race horses in Illinois: a five-year experience.

In order to protect the integrity of horse racing in Illinois, a complex testing of urine and blood specimens collected post-race from winning and special designation horses is continuously conducted. The initial screening by immunoassays was followed by the confirmation on presumptive positive samples. Instrumental screening was also conducted. Perimortem and postmortem specimens and special exhibits (syringes, needles, etc.) were also analyzed. The administration of alkalinizing agents was detected by measuring the total plasma carbon dioxide concentration. The laboratory analyzed specimens collected post race from winning horses and special designation horses at eight race tracks in the State of Illinois over the five-year time period (2004-2009). The total number of specimens collected was 91,808, comprising 45,210 urine specimens and 46,598 blood specimens. The total number of violations was 413 (0.45% of the total number of specimens analyzed); 207 were blood specimens (0.44% of the total blood specimens analyzed), and 206 were urine specimens (0.45% of the total urine specimens analyzed). A total of 220 violations were reported for harness horses, and 193 were reported for Thoroughbred horses. The number of reported violations of the total tested specimens in Illinois was small, but a wide variety of performance-enhancing drugs was shown.

Pyrilamine and O-desmethylpyrilamine detection in equine serum and urine.

Pyrilamine (mepyramine) is an H1-receptor antagonist used in human and veterinary medicine. It has the potential to produce central nervous system effects in horses and therefore may have some impact on an outcome of a horse race. A single oral dose of pyrilamine (300 mg/horse) was given to three animals. Serum samples were collected before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, and 13 days post-administration. Urine samples were collected at 0-1, 1-2, 2-4, 4-6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, 13 days post-administration. Urine and serum samples were initially screened by the pyrilamine enzyme-linked immunosorbent assay (ELISA) kit with subsequent confirmation and quantitation utilizing a newly developed and validated gas chromatography-mass spectrometry (GC-MS) method for pyrilamine and its major metabolite O-desmethylpyrilamine with chlorpromazine as an internal standard. Prior to the basic extraction, urine specimens were hydrolyzed using beta-glucuronidase. The urine extracts as well as the serum samples were then subjected to solid-phase extraction on Bond Elut LRC-PRS columns. Pyrilamine was not found in any of the urine samples but it was present in serum in low concentrations (4-123 ng/mL) up to 6 h after drug administration. The limit of detection and limit of quantitation for the GC-MS method for pyrilamine in serum were 1.5 and 3.1 ng/mL, respectively, and for O-desmethylpyrilamine in urine were 5 and 6.2 ng/mL, respectively. Pyrilamine concentration in serum peaked at 15 min, 30 min, and 1 h in horse #1, #2, and #3, respectively. Urine specimens were screened positive for pyrilamine and its metabolites using ELISA for extended periods of time (4 days in one horse and 9 days in two other animals). Using GC-MS, O-desmethylpyrilamine was detected in urine for 11 days in horse #1, 4 days in horse #2, and 9 days in horse #3. While pyrilamine was eliminated from the bloodstream rather quickly, the metabolite level remained in the urine for days after administration. When evaluating laboratory results, regulators must take into account that a urine sample positive for O-desmethylpyrilamine does not necessarily indicate that the drug remains active in the horse's system, possibly affecting the outcome from the race.

Detection and quantification of low levels of benzoylecgonine in equine urine.

Cocaine (COC) is a highly addictive plant alkaloid expressing strong psychostimulatory effect. It has no medical use in equine veterinary practice. The contamination of the environment with cocaine such as its presence on the US paper currency has been reported few times. There are anecdotal reports of low benzoylecgonine (BE) concentrations (usually much less than 100 ng/mL) being found in urine of race horses. In order to protect horsemen against harsh penalties associated with the presence of trace amounts of BE in horse urine as a result of environmental contamination, in February 2005 the Illinois Racing Board issued new medication rules that established the threshold level of 150 ng/mL for BE in equine urine. The penalties associated with this rule provide for increasing fines ($250, $500, $1000) with successive positive reports against a trainer for levels of BE below 150 ng/mL. A total of 19,315 urine samples were collected over the 2-year period of time from winning horses (both harness and thoroughbred) at race tracks in Illinois for routine drug screening (ELISA). The presence of BE was confirmed by GC/MS in 28 urine samples (0.14%). The concentration range for BE in harness horses (21 detections) was < 5-91 ng/mL, and for thoroughbred (seven detections) was 7-52 ng/mL. To date, the laboratory has not reported concentrations of BE that exceed the established threshold concentration of 150 ng/mL.

An estimate of the proportion of drug-facilitation of sexual assault in four U.S. localities.

In recent years, drugs including flunitrazepam, gamma-hydroxybutyrate, ketamine, and ethanol, have become popularly associated with drug-facilitated sexual assault. Other drugs are also candidates as factors in "drug facilitated sexual assault" (DFSA). The true extent of DFSA is not known, and is difficult to estimate. We recruited sexual assault complainants at four clinics in different parts of the U.S. to anonymously provide urine and hair specimens, and to answer questions about suspected drugging, drug use, and the sexual assault incident. Urine and hair specimens were tested for 45 drugs, including ethanol, and those pharmacologically capable of inducing sedation, amnesia, or impairment of judgment. Analytical test results were used to estimate the proportion of subjects, and the proportion of all complainants to the clinic in the same time period, who were victims of DFSA. Overall, cases of 43% of 144 subjects, and 7% of 859 complainants, were characterized as DFSA. Subjects underreported their use of drugs. The role of toxicological results and history in characterizing DFSA cases is discussed.

Detection of ketamine and norketamine in urine of nonhuman primates after a single dose of ketamine using microplate enzyme-linked immunosorbent assay (ELISA) and NCI-GC-MS.

The general anesthetic ketamine (Ketalar, Ketaject, Vetalar) (KET) is used in human and veterinary medicine for induction of anesthesia for short surgical procedures and routine veterinary examination. Its illicit use by teenagers in rave parties has been reported, and it has recently been identified as a substance associated with sexual assault. One aim of this paper was to study the elimination of KET and its major metabolite norketamine (NKET) in urine collected from five nonhuman primates that received a single dose (5 mg/kg, I.M.) of KET and to study elimination patterns to determine how long after drug administration KET and NKET can be detected. Another aim of this study was to develop and validate a highly sensitive negative ion chemical ionization-gas chromatography-mass spectrometry (NCI-GC-MS) method for the simultaneous quantitation of KET and its major metabolite NKET in urine and to analyze urine samples collected from the animals. The last aim of this study was to apply and evaluate a newly developed ELISA screening methodology for detection of KET and its metabolites in the same urine samples collected from primates which received a single dose of KET. In two monkeys, KET was detected in urine up to 3 days after drug administration (32-7070 ng/mL); in one monkey, it was detected up to 4 days (65-13,500 ng/mL); in one monkey, it was detected only on days 1 and 2 (4000 and 70 ng/mL, respectively); and in one monkey, it was detected 10 days after KET injection (22-35,000 ng/mL). NKET concentrations ranged from 63 pg/mL to 1.75 microg/mL, and it remained in the urine throughout the entire 35-day study period in 4 out of 5 animals. In one monkey, NKET was detected up to 31 days after KET administration. Urine analysis using ELISA revealed that KET and NKET can be easily detectable at 25 ng/mL. In one monkey, KET and its metabolites were detected in urine up to 4 days after drug administration, up to 7 days in two monkeys, up to 11 days in one monkey, and 16 days after KET injection in one monkey. Urine extraction followed by screening using ELISA methodology allowed for significant extension of the detection period in all animals from the study. It is believed that the KET elimination in urine of nonhuman primates is slightly faster than in humans. We propose that NCI-GC-MS be employed to detect NKET as a target compound in urine in toxicological investigations of drug-facilitated sexual assault when KET use by the perpetrator is suspected.

Development of an analytical approach to the specimens collected from victims of sexual assault.

The objective of this study was to develop a screening process for the analysis of sexual assault samples. Recently, the Society of Forensic Toxicologists created a committee to address the issue of drug-facilitated sexual assault (DFSA) in the toxicology field. This committee prepared a list of drugs that could be, or have been, used in DFSAs. The list comprises about 50 compounds, including illicit, prescription, and over-the-counter drugs. Using this list, our laboratory wanted an easy, fast, and sensitive method to analyze a urine sample for all 50 of these drugs. We screened and confirmed for 20 compounds, including cocaine, amphetamines, benzodiazepines, barbiturates, opiates, methadone, alcohol, and PCP. A gas chromatographic-mass spectrometric screening method that was able to detect the remaining 30 compounds following 1 extraction and using only 2 mL of urine was developed. The process is inexpensive and uses equipment available in most forensic toxicology laboratories. This method is recommended for any laboratory that commonly receives specimens collected from sexual assault victims and is interested in a more thorough analysis.

Elimination of 7-aminoclonazepam in urine after a single dose of clonazepam.

The objective of this paper was to determine how long after administration of benzodiazepine clonazepam (CLO), its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in urine collected from 10 healthy volunteers who received a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for urine collection from a victim of drug-facilitated sexual assault in order to reveal drug use. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of CLO and its major metabolite 7-ACLO in urine was developed and validated. The following urine samples were collected from each volunteer: one before CLO administration, and 6 h, and 1, 3, 5, 8, 10, 14, 21 and 28 days after. All urine samples (1 mL) were extracted following addition of the internal standard (D(5)-diazepam) and enzymatic hydrolysis ( beta-glucuronidase) using solid-phase extraction columns. Standard curves for CLO (500-4000 pg x mL(-1)) and 7-ACLO (50-2000 pg x mL(-1)) were prepared by spiking aliquots of negative urine. The urine from every subject was still positive for 7-ACLO 14 days after administration of the drug. Eight of the ten volunteers had measurable amounts of the metabolite 21 days after administration. One volunteer was still positive 28 days after administration. Six of the volunteers had urine concentrations of 7-ACLO that peaked at 1 day after administration. One volunteer had the highest concentration of 7-ACLO at 3 days, two volunteers at 5 days, and one at 8 days. The range of concentrations detected was from 73.0 pg x mL(-1) to 183.2 ng x mL(-1). CLO was not detected in any of the samples.

Analytical developments in toxicological investigation of drug-facilitated sexual assault.

This paper gives a general overview of the drug-facilitated sexual assault phenomenon. Sexual assault perpetrated on both women and men, while incapacitated by so-called date-rape drugs, recently became the focus of many investigations conducted by law enforcement agencies in the US throughout the 1990s; an alarming increase in reports of this crime as well as in the number of scientific publications on drug-facilitated sexual assault has been observed. The list of drugs reportedly associated with sexual assault is long and among others includes flunitrazepam with other benzodiazepines such as diazepam, temazepam, clonazepam, oxazepam, as well as gamma-hydroxybutyrate (GHB), ketamine, and scopolamine. We discuss the most recent analytical developments in the toxicological investigation of drug-facilitated rape designed to reveal drug presence and that may help successfully prosecute perpetrators.

Deposition of 7-aminoclonazepam and clonazepam in hair following a single dose of Klonopin.

The objective of this paper was to determine whether benzodiazepine clonazepam (CLO) and its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in hair collected from healthy volunteers after receiving a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault (DFSA) in order to reveal drug use. Ten healthy volunteers (6 women and 4 men, 23-49 years old) participated in the study. The following hair samples were collected from each volunteer: one before CLO administration, and 1, 3, 5, 14, 21, and 28 days after. All hair samples were pulverized and 50-mg aliquots were sonicated in methanol and digested with 0.1 N HCl at 55 degrees C for 18-24 h. Internal standard, diazepam-d5 (DIAZ-d5) was used. Both extracts were combined and extracted using HCX solid-phase extraction columns. After derivatization with HFBA all extracts were analyzed using highly sensitive negative chemical ionization gas chrometography-mass spectrometry. Standard curves for CLO (20-100 pg/mg) and 7-ACLO (1-20 pg/mg) were prepared by spiking aliquots (50 mg) of negative hair and had correlation coefficients of 0.985 and 0.989, respectively. In addition, two levels of control hair were prepared for CLO and 7-ACLO. All method validation parameters were within acceptable limits. 7-ACLO was detected in hair of 6 out of 10 volunteers. In two cases 7-ACLO appeared in hair three days after CLO intake and remained detectable for the entire 28-day study period (3.6-8.4 pg/mg and 2.7-3.0 pg/mg), and in two subjects it was detectable 21 days later (4.9 and 2.7 pg/mg and 1.2 and 23 pg/mg). In two volunteers 7-ACLO was detected only on day 28 (1.8 and 3.3 pg/mg). CLO was not detected in any of the samples.

The behavioral and cognitive effects of two benzodiazepines associated with drug-facilitated sexual assault.

Recently, sexual assaults have included the use of benzodiazepines to impair the victim. Our aim was to examine the physiological, cognitive, and behavioral effects of flunitrazepam (FN) and clonazepam (CLO). In the first study, ten healthy volunteers received a single oral dose of 2 mg of FN. Mini Mental State Examination (MMSE), behavioral reports and staff observations were then collected. In the second study, ten healthy volunteers received a single oral dose of 3 mg of CLO. Vital signs, performance on the MMSE and Digit Symbol Substitution Test, and behavioral changes were examined. FN significantly decreased systolic and diastolic blood pressure 4 h post drug ingestion with diastolic remaining low at 6 h. CLO was associated with changes in temperature and decreased systolic pressure. FN affected memory and attention 4 h following ingestion. CLO affected memory and attention throughout the study (6 h), and psychomotor performance was decreased 2 h post ingestion. In both studies, subjects were disinhibited and did not perceive their own impairment.